Project Summary Autoimmune diseases, which affect more than 23 million Americans, arise when the immune system targets naturally-occurring proteins (self-antigens), mounting a response against normal tissues and causing a disease state. Current therapies only treat the symptoms of the disease, without curing the underlying autoimmune con- dition; typical treatments are limited to systemic immunosuppressive therapy that can lead to dangerous side- effects. One potential method for generating curative therapies for autoimmune disorders is to induce immune tolerance to the self-antigen that the immune system is aberrantly attacking. This can be achieved using red blood cells (RBCs), which undergo a process where dying RBCs are filtered out in the liver and spleen and are processed in a way that tolerizes the immune system to proteins on or inside the RBCs. However, previous attempts to deliver material to RBCs to tolerize against a specific antigen, such as binding antigens to the RBC surface, led to unwanted immune responses. Another strategy involves hypotonic loading of RBCs, which is time consuming and can process a limited amount of blood, making clinical translation difficult. Ideally, antigens are loaded inside RBCs in a high-throughput, cost-effective manner that does not lead to significant disruption of RBC behavior. We propose to deliver self-antigen proteins to human RBCs to induce antigen-specific T-cell tolerance following the clearance of the antigen-containing RBCs. Our goal is to create off-the-shelf RBC therapies for various au- toimmune disorders by engineering donated O-negative blood with disease-specific antigen in a sterile, rapid, and cost-effective manner. SQZ Biotech has a vector-free microfluidic delivery platform that can deliver any cargo, regardless of size, charge, or chemical structure, to a variety of cell types, including RBCs. The SQZ platform flows cells through a constriction narrower than the cell diameter, temporarily disrupting the cell mem- brane and allowing material diffusion into the cell with little disruption of cell function. Our platform is capable of rapid, high-throughput delivery (>500M cells can be processed in minutes), making the platform amenable to clinical translation. Preliminary data demonstrates that the SQZ platform can deliver antigens to both human and mouse RBCs and that delivery of antigen to mouse RBCs leads to a tolerogenic response in vivo. The ability of the SQZ platform to deliver complex antigen mixtures, such as those generated from digested tissue of the affected organ, could lead to a robust tolerance response precluding antigen identification. We believe that we are uniquely positioned to successfully generate antigen-specific treatment for autoimmune disorders.